REPLI-g Midi Kit

For highly uniform whole genome amplification from small or precious samples

Easy amplification with consistent yields of 40 µg

Unbiased amplification of genomic loci

Reliable results due to Multiple Displacement Amplification (MDA)

Amplified DNA highly suitable for most downstream applications

No risk of DNA degradation during long-term storage

The REPLI-g Midi Kit provides optimized reagents for whole genome amplification (WGA) from small samples using innovative Multiple Displacement Amplification (MDA) technology. The typical DNA yield of a 50 μl reaction is up to 40 μg, with an average product length greater than 10 kb (ranging between 2 kb and 100 kb). Unique REPLI-g technology delivers highly uniform WGA from a variety of small or precious sample types, including purified genomic DNA, or directly from fresh or dried blood, buccal swabs, fresh or frozen tissue, and cells. This simple and reliable method is capable of accurate and unbiased amplification of genomes and generates DNA that can be applied without further purification or quantification for downstream applications that do not require labeling. In contrast to PCR-based WGA technologies, high fidelity rates are increased up to 1000-fold, avoiding costly false positive or negative results.

Genomic DNA samples (10 ng) were denatured using heat (95°C) or the standard REPLI-g Kit alkaline lysis protocol. After amplification using REPLI-g DNA Polymerase the CT values of 2 loci were compared between samples. The low CT values of loci amplified using the REPLI-g Kit alkaline lysis protocol indicate better locus representation, meaning there has been no loss of sequence information at these loci.

Whole genome sequencing of the Bacillus subtilis genome was performed. For analysis, 2 μg of genomic DNA or DNA amplified from 105 cells using the REPLI-g Midi Kit was sheared into 300 bp fragments. For library preparation, 1 μg of each was used. Sequencing was performed on the Illumina MiSeq instrument. [A] Comparable sequence coverage was observed for both gDNA and REPLI-g amplified DNA. [B] Alignment comparison of the genomic loci sequence demonstrates comparably high percentage of alignment for REPLI-g amplified DNA in comparison to the gDNA, which is an indication of minimized levels of junk DNA after WGA (whole genome amplification). Comparison of nonamplified and REPLI-g amplified DNA revealed error rates (mismatch, high-quality error, indels, or chimeras) in a similar percentage range. (Alignment comparison performed using SMALT [Welcome Trust Sanger Institute]).

20 DNA samples amplified using REPLI-g technology, without subsequent DNA purification, were subjected to genotyping analysis using 3 STR loci (CSF1PO, TPOX, and THOI). Results were compared to those obtained for unamplified genomic DNA. The DNA was separated by polyacrylamide gel electrophoresis and visualized by silver staining. A lane with one band represents a homozygote, while a lane with two bands represents a heterozygote for the specific STR locus.

Schematic representation of REPLI-g amplification.

Phi 29 polymerase moves along the DNA template strand displacing the complementary strand. The displaced strand becomes a template for replication allowing high yields of high-molecular–weight DNA to be generated.

The relative representation of 8 loci was determined using real-time quantitative PCR for DNA amplified using [A] REPLI-g technology [B] DOP-PCR and [C] PEP. Locus representation was determined by comparison to 1 µg of unamplified control DNA.

Consistent and accurate whole genome amplification.

Real-time PCR was performed on 47 human loci (2 loci on each autosomal pair, 2 loci on the X chromosome[s], and 1 locus on the Y chromosome) from 44 different samples amplified using REPLI-g technology. Each sample was amplified approximately 10,000-fold with a maximum bias of representation between the loci being only 6-fold.

Uniform DNA yield from various amounts of template.

Various amounts of human genomic DNA were amplified in a standard REPLI-g Midi Kit reaction and aliquots taken at the indicated timepoints. The yield of amplified DNA from a 50 µl reaction was approximately 40 µg, regardless of the amount of starting material.

High yields from a variety of samples, suitable for numerous applications

With the REPLI-g Midi Kit, various clinical and non-clinical research samples can be used, including genomic DNA, fresh or dried blood, fresh or frozen tissue, and cells. Typical DNA yields per 50 µl reaction consistently reach 40 µg (see figure "Consistent DNA yields using any sample type"), while a uniform yield of amplified DNA is usually achieved regardless of the quantity of template DNA (see figure "Uniform DNA yield from various amounts of template"). Obtaining uniform DNA yields from varying template concentrations is always important, but particularly essential for high-throughput applications, which require subsequent genetic analyses to be possible without additional measurement or adjustment of DNA concentration.

The average product length of REPLI-g amplified DNA is typically more than 10 kb, with a range between 2 kb and 100 kb, enabling downstream applications such as complex restriction enzyme analysis and long-range PCR to be carried out. REPLI-g amplified DNA is highly suited for genotyping applications, such as SNP genotyping with TaqMan® primer/probe sets (see figure "Reliable SNP genotyping "), sequencing, and STR/microsatellite analysis (see figure "Accurate genotyping").

Successfully used in next-generation sequencing

Numerous publications have demonstrated the successful utilization of REPLI-g amplified DNA for next-generation sequencing (NGS) applications that range from exome and whole genome sequencing of tumor cells, to metagenomics research, to single cell analysis (for a range of recent publications that successfully used REPLI-g in NGS, please see the WGA resource page). Since the use of whole genome amplified DNA for NGS and array applications has been debated, we detected potential factors that could influence the success of using amplified DNA for these downstream applications. We determined that the quality of input material strongly influences the success of downstream NGS experiments. If working with low quality DNA (e.g., degraded DNA) or aged tissue material, the resulting amplified DNA may not give reliable results (data not shown). However, WGA, using REPLI-g technology, on intact cells or non-degraded purified DNA shows that NGS results are comparable to those obtained with purified gDNA. Sequence coverage and alignment comparison of the genomic loci sequence indicates minimized levels of junk DNA after WGA, whereas error rates are in a similar percentage range for both amplified and genomic DNA (see figure “Comparable NGS (next-generation sequencing) results obtained using purified gDNA or REPLI-g amplified DNA”).

High fidelity whole genome amplification

REPLI-g technology provides highly uniform DNA amplification across the entire genome. Phi29 polymerase can replicate up to 70 kb without dissociating from the genomic DNA template (see figure "Schematic representation of REPLI-g amplification"). In contrast to PCR-based whole genome amplification (WGA) technologies, Phi29 polymerase has 3'→5' exonuclease proofreading activity and maintains up to 1000-fold higher fidelity compared to Taq DNA polymerase during replication. Exonuclease-resistant primers provided in the kit ensure high yields of DNA product, and the WGA buffer system is optimized for very long read length and unbiased locus representation.

REPLI-g outperforms PCR-based WGA methods

Traditional methods of genomic DNA amplification include the time-consuming process of creating EBV-transformed cell lines followed by whole genome amplification using random or degenerate oligonucleotide-primed PCR. Also, PCR-based methods (e.g., DOP-PCR and PEP), as generally used by other suppliers, can produce nonspecific amplification artifacts and give incomplete coverage of loci. In several cases, DNA less than 1 kb long may be generated that cannot be used in many downstream applications. In general, the resulting DNA is generated with a much higher mutation rate due to the use of the low-fidelity enzyme Taq DNA polymerase, which can lead to error-prone amplification that results in, for example, single base-pair mutations, STR contractions, and expansions. In contrast to these disadvantages, REPLI-g provides highly uniform amplification across the entire genome, with minimal locus bias and minimized mutation rates during amplification (see figures "Highly representative amplification using REPLI-g technology" and "Consistent and accurate whole genome amplification").

Principle

Unique REPLI-g technology uses the innovative, high-fidelity enzyme Phi 29 polymerase to amplify complex genomic DNA using Multiple Displacement Amplification (MDA) combined with a gentle alkaline denaturation step to amplify genomic loci uniformly. The typical yield of the REPLI-g Midi Kit is up to 40 µg, and can be easily scaled down according to your needs with the REPLI-g Mini Kit, since both kits are based on the same protocol and use the same reaction volumes. The easy reaction set-up and very low handling time of approximately 15 minutes makes REPLI-g an easy and reliable method to use when complete and unbiased locus representation is needed from limited or precious samples.

Alkaline denaturation of DNA

Genomic DNA must be denatured before use in enzymatic amplification procedures, which is often accomplished using harsh methods such as incubation at elevated temperatures (heat incubation) or increased pH (chemical alkaline incubation). The REPLI-g Midi Kit uses gentle alkaline incubation, allowing uniform DNA denaturation with very low DNA fragmentation or generation of abasic sites. This results in amplified DNA with very high integrity, and maximizes the length of amplified fragments so that genomic loci and sequences are uniformly represented. With the REPLI-g Midi Kit, reliable results without false positive or negative data are ensured in subsequent downstream applications, unlike with other WGA technologies that use heat-induced denaturation that can damage template DNA, leading to biased and underrepresented loci (see figure "Effect of heat and alkaline denaturation on loci representation").

Procedure

Simple, one tube procedure

The REPLI-g Midi Kit uses a simple and reliable method to achieve accurate genome amplification from small quantities of isolated target genomic DNA, or directly from whole blood, dried blood cards, buffy coat, and tissue culture cells (see figure "REPLI-g Mini and Midi procedure). The addition of lysis buffer, which both lysis the sample material and denatures the DNA, is followed by a short incubation (see figure "REPLI-g Mini and Midi procedure"). After neutralization, master mix (including REPLI-g Midi DNA Polymerase) is added and the isothermal amplification reaction proceeds overnight at
30°C. REPLI-g amplified DNA can be stored long-term at –20°C with no negative effects (see figure "Consistent long-term stability").

Specifications for the wide range of REPLI-g Kits

REPLI-g Single Cell

REPLI-g Mini

REPLI-g UltraFast Mini

REPLI-g Midi

REPLI-g Screening

REPLI-g FFPE

REPLI-g Mitochondrial DNA

Starting material

Single cells, gDNA

Purifed gDNA, blood, cells

Purifed gDNA, blood, cells

FFPE tissue, purified gDNA from FFPE tissue

Purified gDNA

(Protocols for other starting materials available from www qiagen.com)

Input amount

Single cells, 2–1000 cells, tissue, purified gDNA (1–10 ng)

>10 ng gDNA, 0.5 µl blood or cells (>600 cells/µl)

>10 ng gDNA, 0.5 µl blood or cells (>600 cells/µl)

Section (1 cm diamter, 10–40 µm thick); >100 ng gDNA

>1 ng purified gDNA

Yield (µg/reaction)

40

10

7–10

40

8

Standard yield: ≤10; High yield: ≤40

3–5

Reaction time

8–16 h

10–16 h

1.5 h

8–16 h

12–16 h

Standard yield: 4 h; High yield: 10 h

8 h

Hands-on time

15 min

15 min

15 min

15 min

15 min

40 min

15 min

Format

Tube

Tube

Tube

Tube

Plate

Tube

Tube

Applications

REPLI-g amplified genomic can be used in a variety of downstream applications, including: